REVIEW

Japanese Encephalitis: A Persistent Threat

Aditi Singh • Shailendra K. Saxena •

Apurva K. Srivastava • Asha Mathur

Received: 9 September 2011 / Accepted: 14 November 2011 / Published online: 18 January 2012

� The National Academy of Sciences, India 2012

Abstract Japanese encephalitis virus (JEV) belongs to

arthropod-borne virus family, which are infectious agents

transmitted by blood-sucking arthropods. They multiply in

the tissues of the arthropod without evidence of disease or

damage. The principal vector is Culex mosquito, most

important being C. tritaenorhynchus, which is a rural mos-

quito, breeding extensively in rice fields and bites large

domestic animals. In temperate zones, this vector is present

in greatest density from June to November. The natural cycle

of JEV consists of pig–mosquito–pig or bird–mosquito–bird

cycle. Pigs serve as most important biological amplifiers and

reservoirs. JEV is transmitted to humans through the bites of

infected mosquitoes. Humans are accidental, dead-end hosts

as they do not develop a level of viraemia sufficient to infect

mosquitoes. The infection with JEV ranges from nonspecific

febrile illness to a severe meningoencephalomyelitis illness.

The risk for Japanese encephalitis varies by local ecology

and season. In temperate areas of Asia, human disease

usually peaks in summer and falls down after that and these

seasonal epidemics can be explosive with thousands of cases

occurring over a period of several months; whereas, in the

subtropics and tropics, transmission can occur throughout

the year, with disease at a peak during rainy season. A

detailed study has been done on geographic distribution of

JEV and incidence and burden of disease in India. Vacci-

nation proves to be the best measure to protect the individual

against any disease. So, large scale immunization of sus-

ceptible human population is highly important to prevent

this deadly infection. In India, vaccinations against Japanese

encephalitis are administered in areas where the disease is

hyperendemic.

Keywords Japanese encephalitis � Encephalopathy �Mosquito-borne infection

Introduction

Japanese encephalitis (JE), one of the most common cause

of acute encephalitis in tropics [1, 2], has generated con-

siderable public anxiety in India. Caused by a neurovirulent

RNA flavivirus, JEV and transmitted by mosquitoes, the

occurrence of this disease highlights the inter relationship

between man and animals, birds and the environment.

Among an estimated 35,000–50,000 cases reported annu-

ally, approximately 20–30% of patients die and 30–50% of

survivors suffer from neurological sequelae [3, 4]. The

infection with JEV ranges from nonspecific febrile illness

to a severe meningoencephalomyelitis illness [5]. The

symptoms are serious and no definite treatment is available.

JE is mainly a disease of children, but it can occur among

persons of any age which is mostly travel related and is

generally very rare [6–8]. Travellers living for prolonged

periods in rural areas where JE is endemic or epidemic are

at greater risk than short term and urban travellers.

Vaccination proves to be the best measure to protect the

individual against any disease. Hence, large scale immuni-

zation of susceptible human population is highly important

A. Singh

Amity Institute of Biotechnology, Amity University Lucknow,

MOC Campus, Near Malhaur Crossing, Lucknow 226010, India

S. K. Saxena

Centre for Cellular and Molecular Biology (CSIR), Uppal Road,

Hyderabad 500007, India

A. K. Srivastava � A. Mathur (&)

Department of General Pathology & Microbiology, Saraswati

Dental and Medical College, Tiwariganj, Faizabad Road,

Lucknow 227105, India

e-mail: asha.mathur@gmail.com

123

Proc. Natl. Acad. Sci. Sect B. Biol. Sci. (January–March 2012) 82(1):55–68

DOI 10.1007/s40011-011-0005-x

to prevent this deadly infection. In India, vaccinations

against Japanese encephalitis are administered in areas

where the disease is hyperendemic, like Gorakhpur region of

Uttar Pradesh [9]. In the case of JE, it is essential to

immunize the pigs (amplifying host) also to interrupt the

transmission of disease [10]. Thus, control of vectors and

intense immunization in endemic regions are the only

effective preventive measure. The vaccines available for JE

these days are of three types—(i) A Mouse-brain derived

inactivated vaccine (only vaccine which is WHO approved),

(ii) cell-culture derived inactivated JE vaccine (which was

developed in China), and (iii) cell-culture derived live

attenuated JE vaccine (which is the most cost-effective

vaccine [11]). Apart from them, recombinant protein based

vaccine and DNA vaccines are still in development process.

Historical Background

Japanese encephalitis was first recognised as a clinical

entity in Japan in 1871; but the causative agent (JEV) was

later isolated from a fatal human case in 1934 in Japan and

was serologically and virologically established as a proto-

type (Nakayama) strain [12]. The Nakayama strain was

later used in the development of mouse brain inactivated

virus vaccine. It took almost 25 years after its first

isolation, to elucidate the mode of transmission by mos-

quito vector. In last three decades, the focus of viral epi-

demics has changed from temperate zone of Asia to South

and Southeast Asia [13]. In India, acute viral encephalitis

was first detected in 1955 and since then, it has taken away

thousands of lives. Several encephalitis outbreaks reported

in the later years remained undiagnosed.

Ecology and JEV Transmission

JEV belongs to arthropod-borne virus family (arboviruses),

which are a group of infectious agents that are transmitted

by blood-sucking arthropods. They multiply in the tissues

of the arthropod without evidence of disease or damage.

The principal vector of this zoonotic disease is Culex

mosquito, most important being C. tritaenorhynchus, oth-

ers are C. vishnui and C. pseudo-vishnui [14, 15], which are

primarily rural mosquitoes breeding extensively in rice

fields and preferentially bite large domestic animals like

pigs and wading birds [16]. In temperate zones, this vector

is present in greatest density from June to November. The

natural cycle of JEV consists of pig–mosquito–pig or bird–

mosquito–bird cycle (Fig. 1). Other minor hosts are cattle,

buffaloes, goats, sheep, horses, rodents, monkeys, dogs and

bats [17].

Fig. 1 Transmission cycle of

Japanese encephalitis virus

(JEV). JEV is transmitted in an

enzootic cycle between Culexmosquitoes and amplifying

vertebrate hosts, primarily pigs

and wading birds like water

fowls and egrets. Human beings

serve as a dead-end host in the

JEV transmission cycle with

low levels of viremia. Humans

play no role in the maintenance

or amplification of JEV, and the

virus is not transmitted directly

from person to person

56 Proc. Natl. Acad. Sci. Sect B. Biol. Sci. (January–March 2012) 82(1):55–68

123

Pigs are the most important biological amplifiers and

reservoirs [18]. Countries, like Pakistan, that do not rear

pigs experience rare JE cases [19]. In the study done on

rural children in Thanjavur district (Tamil Nadu), an area

with extensive paddy cultivation, JE occurrence was found

to be very low and without epidemics. The reason could

possibly be high cattle to pig ratio (400:1) [20]. JEV is

transmitted to humans through the bites of infected mos-

quitoes. Humans usually do not develop a level of viraemia

sufficient to infect mosquitoes [21]. Therefore, humans are

accidental, dead-end hosts, and human JE cases imported

into non-endemic areas represent a minimal risk for sub-

sequent transmission of the virus [22]. Direct person-to-

person spread of JEV does not generally occur until and

rarely it is through intrauterine transmission [23, 24].

Blood transfusion and organ transplantation may also serve

as potential mode of JEV transmission [25]. The risk for JE

varies by local ecology and season. In temperate areas of

Asia, human disease usually peaks in summer and falls

down after that [26–28] and these seasonal epidemics can

be explosive with thousands of cases occurring over a

period of several months; whereas, in the subtropics and

tropics, transmission can occur throughout the year, with

disease at a peak during rainy season. A recent study in

China has correlated the different weather variables like

temperature, relative humidity as possible predictors of

Japanese encephalitis incidence for regions with similar

geographic, weather and socio-economic conditions [29].

However in endemic areas (e.g., southern India) sporadic

cases may occur throughout the year due to congenial

climatic conditions [30].

Geographic Distribution of JE

Japanese encephalitis occurs throughout Asia and parts of

the Western Pacific areas (Fig. 2). The disease was rec-

ognized in the first half of 20th century in the temperate

areas of Asia, mainly Japan, Korea, Taiwan and mainland

China [31–33]; but over the past few decades, the disease

appears to have spread to Southeast Asia, India, Bangla-

desh, Sri Lanka and Nepal [34–37]. By 1990s, JEV had

spread to non-Asian regions, Saipan and Australia, where it

was reported first from Torres Strait islands and then from

northern mainland [38, 39]. The reasons for this wide

distribution can be population shifts or changes in climate,

ecology, agricultural practices, animal husbandry or

migrating bird patterns [40]. Though exact cause is

uncertain, these factors could very well contribute to fur-

ther spread, potentially beyond Asia and Western Pacific.

Incidence and Burden of Disease in India

In India, the disease has been recognized since 1945 and

though virus causes epidemics, it is endemic in several

parts of the country [41–44]. The earliest evidence of the

prevalence of JE virus was cited through serological

Fig. 2 Approximate

geographic range

(epidemiology) of Japanese

encephalitis highlighting the

areas which are engulfed by

Japanese encephalitis virus

Proc. Natl. Acad. Sci. Sect B. Biol. Sci. (January–March 2012) 82(1):55–68 57

123

studies carried out at the Virus Research Centre, Pune in

1952 and then in 1956, the isolation of JE virus was made

from the wild caught mosquitoes, establishing its presence

in India. However, the virus could not be recovered from

human until 1958, when three isolations were made from

the brain tissue of encephalitis cases in Tamil Nadu [45].

The geographic area affected by JEV has expanded in the

last 60 years with higher epidemic activity in North India

and Central India (Fig. 3). Since the confirmation of first

clinical case of JE from Vellore in 1955 [46], there had

been many major JE outbreaks reported from Bihar, Uttar

Pradesh, Assam, Manipur, Andhra Pradesh, Karnataka,

Madhya Pradesh, Maharashtra, Tamil Nadu, Haryana,

Kerala, West Bengal, Orissa and Union Territories of Goa

and Pondicherry [47, 48].

Carey et al. [49] had reported almost 65 JE cases from

South India from 1955 to 1966 by demonstrating specific

neutralizing antibodies to JE. In its first major outbreak in

West Bengal, JE had taken about 300 lives from the two

districts, Burdwan and Bankura in 1973 [50]. During 1978,

several outbreaks of JE occurred in different parts of India

viz. Kolar district of Karnataka, Tirunelvelli, Benpura,

Burdwan and adjoining areas of West Bengal, Dhanbad

district of Bihar, Dibrugarh district in Assam, Goa on the

west coast [42] and eastern districts of Uttar Pradesh [41],

which was one of the worst JEV outbreaks. From this

outbreak, a successful isolation and identification of JE

virus as the causative agent was made from the brain tissue

of a fatal case of encephalitis from Gorakhpur district for

the first time by Asha Mathur and her team at the King

Georges’ Medical College, Lucknow [41]. Outbreak of JE

was again reported in 1981 from South Arcot district [46]

and from Gorakhpur in 1988 with as much as around 875

cases reported [51]. Eastern paddy growing districts of

Haryana suffered an epidemic of encephalitis in 1990 [43]

and Muzaffarpur district of Bihar in 1995. JE had emerged

as a major public health problem in naive non-endemic

area like Kerala [52], where a large outbreak of JE

occurred in Kuttanad area of Allepey district [53]. In a

severe epidemic of JE during October–November 1999 in

Fig. 3 Approximate display of

JE occurrence areas in India.

Different colours in the map

highlight the intensity by which

JEV occurs in different areas

58 Proc. Natl. Acad. Sci. Sect B. Biol. Sci. (January–March 2012) 82(1):55–68

123

Andhra Pradesh, out of 23 districts, fifteen districts were

affected with 873 cases and about 20% mortality [54].

Another outbreak occurred in July 2003 in Warangal and

Karim Nagar district of Andhra Pradesh [55]. From north-

east regions, JE outbreaks which were reported from

Assam during July–August 1989, had affected a population

of *36,000 with 50% case fatality rate [56]. After that,

several other outbreaks have been reported from Assam

between 2000 and 2002 [57].

Thus, encephalitis by JEV that was known to cause

epidemics in several parts of the country now has become

endemic in many parts of India, especially in South India

[58] (including Tamil Nadu [47]) and terrain belt of Uttar

Pradesh [59], which is a matter of serious public health

problem. In Uttar Pradesh, the most severely JE affected

region is Gorakhpur and the longest epidemic in three

decades, had been reported from this district from July to

November, 2005, in which 5,737 people were affected and

approximately 1,344 died [60]. More recent studies on

phylogeny of virus revealed that the epidemic was caused

by JEV GP78 [61]. Gorakhpur and surrounding region

in Uttar Pradesh still gets consistent outbreaks of fatal

acute encephalitis syndrome (AES), of which *10–15% is

caused by JEV [62].

Among children in endemic zone, the incidence of

laboratory confirmed JE varies widely areawise, the range

being 5–50 cases per 100,000 children/year [28, 63]. The

number of affected persons is magnified also because of

many non-JE cases with unidentified etiologies. So, now

the cases are classified as JE and non-JE and enterovirus 89

and 76 were isolated from the CSF of non-JE encephalitis

cases from Gorakhpur in 2006 [11].

Japanese Encephalitis Virus

Four Flaviviral infections, of which three are encephalitis,

are transmitted by mosquitoes from birds [64]. Clinically

the most serious of these infections is ‘‘Japanese enceph-

alitis’’, caused by Japanese encephalitis virus (JEV). The

North Indian strain of Japanese encephalitis virus (JEV) is

GP78, which is phylogenetically closer to the Chinese

SA14 isolate. The other, Vellore P20778 isolate, was

obtained from southern India in 1958 [65]. JEV is an

enveloped virus and has a linear, single stranded, positive-

sense *11 kb long RNA as a genome and measures

40–50 nm in diameter with a nucleocapsid which is sur-

rounded by a lipid envelope (Fig. 4). The genomic RNA

Fig. 4 Morphology of the Japanese encephalitis virus and the detailed display of the organisation of viral genome highlighting the structural and

non-structural genes and also the structure of protein extracted

Proc. Natl. Acad. Sci. Sect B. Biol. Sci. (January–March 2012) 82(1):55–68 59

123

contains a single open reading frame (ORF) and codes for a

polyprotein of *3,400 amino acids. This polyprotein is

cleaved by viral and host proteases into ten proteins: three

structural proteins (capsid, M and E proteins) and seven

non-structural proteins [66] (NS1, 2A, 2B, 3, 4A, 4B & 5).

Envelope or E defines the type specificity. The viral RNA

has non-coding regions of 95 and 585 bases at 50 and 30

ends which interact with viral or host proteins which are

important for virus replication [67]. Looking at the regular

epidemics of JE, studies are being conducted to look for

any mutation with the viral genome and as demonstrated by

Pujhari et al. [68] a novel mutation was detected in domain

II of the envelop gene of JEV circulating in North India.

More studies are required to ascertain its role in JE

pathogenesis.

Genotypes of Japanese Encephalitis Virus

Epidemics of encephalitis were described in Japan from

1870s onwards and have subsequently been found across

most of Asia [3]. For most of the major human pathogens,

we have little idea about where they have originated.

Because JEV has so rapidly spread to new areas, tracing its

geographical origin has been somewhat possible [69] and

phylogenetic comparison with other flaviviruses suggest

that it evolved from an ancestral virus a few centuries ago.

Five genotypes of JEV have been identified based on

nucleotide sequences of C/PrM and E genes: Genotype 1 to

5 [70]. Among the five, GIV and GV appeared rarely.

Genotype 1 (GI) is found to be circulating in northern

Thailand, Cambodia and Korea. It has been less associated

with human disease than genotype 3 in China; but a recent

study reported isolation of genotype 1 (GI) from a vacci-

nated boy who developed JE [71]. Genotype 2 (GII)

includes isolates from southern Thailand, Malaysia, Indo-

nesia and Northern Australia. Isolates from temperate

regions of Asia i.e. Japan, China, Taiwan, Philippines,

Vietnam, Sri Lanka, Nepal and India has been put in

Genotype 3 (GIII), whereas GIV includes isolates from

Indonesia only [72]. GV included only a single strain iso-

lated from a patient from Mummer, Malaysia in 1952 [73].

Since all five genotypes have been found in the Indonesia

Malaysia region, it is likely that these current five geno-

types evolved around the same region (Fig. 5). GI and GIII

occur in epidemic regions, whereas GII and GIV are

associated with endemic disease [3].

JEV is naturally transmitted between vertebrate hosts by

Culex and other mosquitoes. The reasons for its spread are

uncertain but many include changing agricultural practices,

such as increasing irrigation and animal husbandry [74].

JEV continues to spread with recent outbreaks in India,

Nepal and Australia. GIII is most widely distributed and

was the only genotype found in India till 2007 [62].

However, in a recent study done by Fulmali and his co-

workers demonstrated simultaneous detection and isolation

of GI and GIII JEV strains from 66 acute encephalitis

syndrome (AES) patients. Clinical syndrome for the two

strains was indistinguishable. Thus GI and GIII detection

indicates their co-circulation and association with human

infections in Gorakhpur region [9]. GI isolate from India

has been found to share close relationship with GI strain

from Japan and Korea. GIII was the major genotype cir-

culating in Japan, Korea and Vietnam until the early 1990s.

However, by mid 1990, G III disappeared in Japan and

Vietnam and GI supplanted it. This phenomenon is called

‘‘Genetic Shift’’ [75]. In Japan, JEV has been shown to

overwinter and settle [76]. In India, the exact mode of

introduction of GI JEV is not clear. It is possible that it may

have been introduced into India through migratory birds

like other Asian countries [77]. More studies are required

to establish the role of migratory birds in JE transmission.

Pathology and Pathogenesis

A considerable degree of variation exists in neurovirulence

and peripheral pathogenicity among JE virus strains and

the mechanism of JEV pathogenesis is still unclear [78]. It

has also been noted that typical pathological changes

associated with each epidemic varied dramatically [79].

After the virus gets inoculated into the skin by the bite of

an infected mosquito, it is quickly disseminated in the body

and proliferates in the reticuloendothelial system. There is

a transient period of viremia after which invasion of the

central nervous system occurs. JEV is thought to invade

brain via vascular endothelial cells by endocytosis [80],

which involve clathrin and membrane cholesterol, referred

to as lipid rafts acting as portals for virus entry [81]. In

brain, the distribution of virus is in the thalamus, hippo-

campus, substantia nigra and medulla oblongata [82]. In

the neurons, JEV replicates and matures in the neuronal

secretary system. Neurocysticercosis (NCC) in 33% of

patients dying with JE has been seen, but in only 1% of

patients dying due to other causes, suggesting that NCC

may somehow predispose to JE [79, 83]. A high level of

tumour necrosis factor is correlated with adverse outcome

in JE [84].

Clinical Spectrum

The majority of human infections with JEV are asymp-

tomatic with proportion of apparent to inapparent JEV

infection found to be 1:300 to 1:1000 during epidemics

[85]. In a study of JE infection in Assam, the ratio of

60 Proc. Natl. Acad. Sci. Sect B. Biol. Sci. (January–March 2012) 82(1):55–68

123

apparent to inapparent infection was shown to be 9.1:100

[86]. Milder forms of disease (e.g. aseptic meningitis or

undifferentiated febrile illness) can also occur, but have

been reported more commonly among adults [87]. JE

affects all age groups, with higher incidence in children

aged \15 years of age [37, 88]. However, in countries

(China and Japan) with childhood JE immunization pro-

grammes, the overall incidence of JE has decreased and

similar numbers of cases are observed among children and

adults [89, 90]. Because unvaccinated travellers from non

endemic countries usually are immunologically naive,

travel associated JE can occur in persons of any age [22].

Among patients, who develop clinical symptoms, the

incubation period is 5–15 days. Table 1 shows the course

of disease which is divided into three stages—prodromal

stage, acute encephalitic stage and late convalescent stage.

Fatality in JE patients is usually seen within 24–48 h of

admission [91] and among the survivors; at least one in 200

affected individuals develop severe psychoneurological

sequelae [92] in the form of parkinsonism [93], convulsive

disorders, motor abnormalities, impaired intellect, hearing

deficit, scholastic backwardness, speech disturbances [94]

and other subtle neurological signs. Movement disorders

have also been observed [95]. The disease can be much

more difficult to detect in infants.

Immune Mechanism Against JE Infection

After the entry, JE virus is partially destroyed at the site of

its entry and the remaining virus is disseminated by local

and systemic extra neural replication leading to viraemia.

After primary infection with JEV, presence of IgM anti-

bodies and T-lymphocytes are seen until about 2 weeks

[96, 97]. But antibodies alone are neither capable of ter-

minating the viraemia nor preventing the subsequent

infection [98]. Pregnancy is known to cause immunosup-

pression and persistent maternal infection or pregnancy

induced reactivation of the virus may cause foetal infec-

tion. Isolation of JEV from human placenta and foetuses

has been reported by Chaturvedi et al. [23], whereas

Mathur et al. [24] have described an animal model of

Fig. 5 Sequence phlylogeny of JEV isolates. Sequence phylogeny of

Japanese encephalitis virus isolates from Gorakhpur (India) 2005

epidemic, with reference to other Southeast Asian isolates and

flaviviruses based on partial E gene sequence. The tree was generated

by neighbor-joining method. Bootstrap values are indicated at the

branch points. DEN, WNV, KUN, SLEV and MVE denotes Dengue

virus, West Nile virus, Kunjin virus, Saint Louis encephalitis virus

and Murray Valley encephalitis virus respectively [61]

Proc. Natl. Acad. Sci. Sect B. Biol. Sci. (January–March 2012) 82(1):55–68 61

123

congenital infection with JEV, showing an active replica-

tion of virus in placenta. They have also shown transpla-

cental transmission of virus during consecutive pregnancies

of mice. JEV can establish latency within different organs

despite the presence of antiviral antibodies [99]. Immu-

nological and virological evidence for persistence of JEV

have been described in human nervous system also [100].

Latent and recurrent infection as a consequence of acti-

vation of latent JEV has been reported in humans [101].

A significant decrease in serum iron levels, a frequent

feature of microbial invasion is observed during JE infec-

tion [102, 103]. An early influx of macrophages followed

by neutrophils at the site of injury in different organs in

humans [104] and mice [105] has been reported, which is

correlated with the production of a neutrophil chemotactic

macrophage derived factor, MDF [106], with development

of hypoglycaemia [107, 108]. This chemotactic protein

(MDF) has been shown to play a protective role in the host

defence against JEV, through production of reactive

oxygen intermediates [109] in neutrophils and reactive

nitrogen oxide species [110] degrading the virus protein

and RNA. In other studies, mechanism of proinflammatory

cytokine [111] and cytokine secretion at molecular level

has been studied and the involvement of monocyte and

macrophage receptor, CLEC5A, in severe inflammatory

response in JEV infection of brain is reported [112]. Misra

et al. [113] have found increased levels of proinflammatory

and anti-inflammatory cytokines and monocyte chemoat-

tractant protein-1 in the serum of rats after JEV infection.

The mechanism of JEV pathogenesis is still unclear [78].

For the development of appropriate and effective therapy

there is an immediate requirement to understand the role of

host factors in JEV-induced neuropathogenesis [114].

Laboratory Diagnosis

Clinical findings with JE are nonspecific and might include

moderately elevated white blood cell count, mild anaemia

and hyponatremia [22, 115]. The clinical suspicion of

encephalitis can be supported with CSF analysis [30, 115].

Usefulness of various conventional magnetic resonance

imaging (MRI) of the brain is better than computed

tomography for detecting JEV associated changes [116].

Recently the utility and usefulness of various conventional

MRI sequences in the diagnosis of acute viral encephalitis

has been evaluated. Viral encephalitis patients were

assessed by clinical evaluation and confirmation was done

by analyzing serum or CSF for dengue, JE, herpes, mea-

sles, echo, coxsackie and polio viruses using ELISA or

PCR. It was found that the MRI changes are correlated with

type of encephalitis and duration of illness [117].

In India, the actual JE burden could be estimated only by

strengthening diagnostic facilities for JE confirmation in

hospitals [10]. The laboratory diagnosis of a confirmed case

of Japanese encephalitis is based on either isolation of virus

from specimen or by antibody detection. Because humans

have low or undetectable levels of viremia by the time dis-

tinctive clinical symptoms are recognized, virus isolation

and nucleic acid amplification tests (NAAT) are insensitive

and should not be used for ruling out a diagnosis of JE [22].

For rapid diagnosis of JE, detection of antigen in CSF using

immunofluorescence [118] and reverse passive haemag-

glutination [119] using poly or monoclonal antibodies has

been successful. The reverse transcriptase polymerase chain

reaction amplification of viral RNA may help in rapid

detection of JEV in samples [13].

Acute phase specimens are tested for JEV-specific IgM

antibodies using a capture enzyme-linked immunosorbent

assay (MAC-ELISA) [22]. Many modifications of MAC-

ELISA such as nitrocellulose membrane based IgM capture

dot enzyme immunoassay (Mac DOT), Avidin biotin system

(ABC Mac ELISA), biotin labelled immunosorbent assay to

Table 1 Clinical picture of JEDisease course Signs and symptoms

Prodromal stage General malaise, fever, anorexia, headache, vomiting. Abdominal pain

& diarrhoea common in children

Acute encephalitic stage Continuous high fever, nuchal rigidity, seizures, muscle weakness,

speech, hearing & vision problems, partial paralysis, altered

sensorium leading to coma

Late convalescent stage Complete recovery in mild cases, but neurological sequelae in severe

cases

Table 2 Vaccines available against Japanese encephalitis virus

Three types produced and used worldwide:

Formalin-inactivated mouse brain derived—produced from

Nakayama strain, safe and effective, most widely used. But

expensive, complicated dosing schedule and side effects

reported

Inactivated hamster kidney cell vaccine—developed from Beijing

P3 strain in China, has efficacy of *75–90% with few side

effects.

Live attenuated hamster kidney cell vaccine—developed from SA

14-14-2 strain by China, has shown high efficacy with single

dose and is cost effective. Being successfully used in India,

Japan, Korea, Thailand etc.

62 Proc. Natl. Acad. Sci. Sect B. Biol. Sci. (January–March 2012) 82(1):55–68

123

sandwich ELISA and antibody capture radioimmunoassay

(ACRIA) have been successfully tested for antibody detec-

tion [13]. Therefore, a positive IgM test has good diagnostic

predictive value, but cross-reaction with arboviruses from

same family can occur. The performance of three commer-

cial JEV IgM antibody capture ELISA (MAC ELISA) kits

has been evaluated with acute encephalitis specimens from

India and Bangladesh and were found to be having high

specificity (95–99.5%), but low sensitivities (15–57%)

[120]. The two commercially available kits in India: Japa-

nese encephalitis—Dengue IgM combo ELISA (Panbio

Diagnostics) and JEV-Chex IgM capture ELISA (Cyton

Diagnostics Limited) for the diagnosis of Japanese

encephalitis in field samples have also been compared

recently, in which the sensitivity and specificity were found

to be 63.6 and 98.4% respectively for XCyton ELISA and 75

and 97.7% for Panbio ELISA [121].

Treatment and Management

There is no specific treatment or specific antiviral agent or

other medication to mitigate the effects of JEV infection

[122]. Thus it becomes highly important that the infection be

managed carefully. Only one in every six deaths is directly

due to JE virus and five out of six are preventable with

prompt and early management, thus bringing down the case

fatality rate of JE from 35–50% to less than 6% [30]. In

different studies, monoclonal antibodies [13], corticoste-

roids, interferon alpha-2a or ribavirin have not been shown

to improve clinical outcome [22]. Swarup et al. [123] have

shown the effect of rosamarinic acid (RA) as an efficient

antiviral agent that reduced JE viral load along with proin-

flammatory cytokines in experimental animals. Therapeutic

role of diethyldithiocarbamate (Anti JEd) have been dem-

onstrated during JEV infection [124]. Nazmi et al. [125]

have demonstrated an effective way to counter the virus

through inhibiting viral replication by using anti-sense

molecules (Vivo-Morpholino) directed against the viral

genome. Mycophenolic acid has been found to inhibit the

replication of JEV in vitro and protected mice in vivo [126].

Outcome and Sequelae

JE has a case-fatality ratio of approximately 20–30% [22,

37, 123] and although some motor deficits improve after

acute illness, 30–50% survivors have neurologic or psy-

chiatric sequelae for years [123, 127]. Drug therapy and

physiotherapy are necessary for survivors with sequelae

like seizures, paralysis, movement disorders, Parkinsonism

and psychiatric problems [30]. Also recurrence of symp-

toms after recovery with fresh neurologic deficits can occur

as the virus remains latent in peripheral lymphocytes, and

can later cause recurrence [99].

Prevention and Control

Preventive measures are very important for minimizing JE

infection. Vector control should include measures such as

eliminating potential mosquito breeding areas, shifting

pigsties and environmental sanitation. Reduction of

breeding source for larvae can be done by water manage-

ment system with intermittent irrigation system, which is

nothing but a strategy of alternate drying and wetting water

management system in the rice fields. Equine and swine in

affected areas should be vaccinated [10]. For humans in

endemic areas, vaccination should be implemented, as well

as personal protective measures. Vector control alone

cannot be relied upon to prevent JE. Rapid identification

and diagnosis is important for protecting the public and our

livestock from this disease. For the disinfection, Biosafety

level three precautions and practices are recommended for

investigators working with this virus [128].

Vaccination

In the last five decades, JE has been effectively controlled

and virtually eliminated in Japan, Taiwan, China and Korea

after implementation of childhood immunization programs

using inactivated mouse brain derived vaccine [129]. In

Cambodia, JE surveillance commenced in 2006, and an

immunization program has been planned [130]. Therefore,

vaccination of the population at risk against JE, appears to

be the most important control measure now, and in the

future. At present, three types of vaccines are in use

worldwide (Table 2 ).

(i) Formalin-inactivated mouse brain derived vaccine is

the only WHO approved vaccine. Asian field trials

have shown that two doses (1–2 weeks apart) provide

an efficacy of 91% [131]. However, three doses (0, 1

and 6 months) are being followed to immunize

children in endemic zone. The vaccine is contraindi-

cated for women who are pregnant and those who are

immunocompromised. It is also being produced from

Beijing-1 strain due to its high cross-reactivity among

the JEV strain. The vaccine is being manufactured by

Research Institute, Kasauli in India [132]. Vaccine has

some common allergic reactions like fever, headache,

malaise, dizziness, erythema, swelling and tenderness

[133]. The mucocutaneous and neurologic allergic

reactions reported after JE vaccination, may vary in

frequency, and therefore these should be evaluated

Proc. Natl. Acad. Sci. Sect B. Biol. Sci. (January–March 2012) 82(1):55–68 63

123

very carefully yearly [133]. Though safe and effective,

the vaccine has limitations in terms of cost, availabil-

ity and lack of long term immunity.

(ii) Inactivated primary hamster kidney cell derived

vaccine has been developed in China. The efficacy

was found to be 76–90% in a field trial in China [134]

and there were fewer side effects. A vero-cell culture

derived inactivated vaccine from attenuated SA

14-14-2 strain has been developed, which induced

high titers of neutralizing antibodies in experimental

animals [135]. Another vero cell-culture derived

inactivated JE vaccine using Indian isolate (P

20778) has been developed. It generated high titers

of anti-JEV antibodies in mice [136].

(iii) A cell-culture derived live, attenuated SA 14-14-2

vaccine from China has recently become interna-

tionally available and is increasingly replacing the

inactivated, mouse brain-derived vaccine in Asia. A

single dose of the vaccine demonstrated 96%

efficacy after 5 years [137]. The vaccine is very

cost-effective and appears to be safe and neuroat-

tenuation is stable [13]. The government of India

introduced the SA 14-14-2 vaccine in 2006, and

nearly 50 million children 1–15 years of age have

been reached through vaccination campaigns and

routine immunization [138].

JE vaccine candidates in late-stage of development

include a live, attenuated chimeric virus vaccine (IMO-

JEV�) and an inactivated Vero cell-derived vaccine; each

based on the SA 14-14-2 strain. IMOJEV� (previously

known as ChimeriVaxTM

—JE), is a novel recombinant chi-

meric vaccine, developed using the yellow fever virus

(YFV) vaccine vector as its backbone. It is found to be safe,

highly immunogenic and capable of inducing long-lasting

immunity in both preclinical and clinical trials [139].

Additionally, 2 inactivated, Vero cell-derived vaccines

based on the Beijing-1 strain are being developed in Japan

[140]. Second generation recombinant vaccines are also

being developed, where signal sequences of PrM with genes

encoding PrM and E proteins are packaged into vectors, e.g.

E. coli and vaccinia [141]. Studies on plasmid DNA-based

JEV vaccine are also giving promising results on experi-

mental animals [142] and in one of the study; the JEV

candidate DNA vaccine was capable of generating protec-

tive levels of neutralizing antibodies in rhesus monkeys

[143]. DNAzymes (Dzs) that cleave the RNA sequence of

the 30-NCR of JEV genome in vitro, on intra-cerebral

administration in JEV-infected mice led to more than

99.99% inhibition of virus replication in brain, resulting in a

dose-dependent extended life-span or complete recovery of

the infected animals [144]. New vaccine development, along

with progress in surveillance and immunization, offers

promise for sustainable control of clinical JE. To achieve

this, global partners are working together to develop a

strategic plan for JE control by 2015 [145].

Future Challenges

Currently in India, since the risk of JE is limited to focal

areas, its immunization is not included in the National

Immunization Program. It is recommended for active

immunization only in JE prone areas [30]. There is a need for

a proper pilot trial on JE vaccination involving hyperen-

demic districts for making a policy on mass JE vaccination

in the community. JE vaccination should be considered in

travelers, who are likely to spend[30 days in a JE-endemic

country or travelers on brief trips if they have extensive

outdoor or night time exposure in rural areas during periods

of active transmission [22, 146]. It is also been reported that

routine availability of vaccine and information, education

and communication strategies play important roles in

improving knowledge and achieving high vaccination rates

[147]. For the development of effective therapy, there is an

immediate requirement to understand the host factors in

JEV-induced neuropathogenesis [109]. Because the live

attenuated JE vaccine used in India is derived from GIII

strain SA14-14-2, vaccine’s efficacy to protect against GI

must be evaluated. Also the expansion of GI JEV into other

parts of India should be continuously tracked [9]. It is also

necessary to elucidate the ecology of migrating reservoir

animals. Genotyping of both the mosquitoes and JEV should

be done concurrently at many locations in temperate and

tropical regions simultaneously. Any distinct biological

markers, like susceptibility of replaced strains in mosqui-

toes, birds etc. should be elucidated.

References

1. Halstead SB, Jacobson J (2008) Japanese encephalitis vaccines.

In: Plotkin SA, Orenstein WA, Offit P (eds) Vaccines, 45th edn.

Saunders Elsevier, Philadelphia 311–352

2. Erlanger TE, Weiss S, Keiser J et al (2009) Past, present, and

future of Japanese encephalitis. Emerg Infect Dis 15:1–7

3. Solomon T, Dung NM, Kneen R et al (2000) Japanese

encephalitis. J Neurol Neurosurg Psychiatry 68:405–415

4. World Health Organization (2006) Sixteenth meeting of the tech-

nical advisory group on immunization and vaccine preventable

diseases in the Western Pacific Region; Manila, Philippines. Avail-

able from http://www.wpro.who.int/NR/rdonlyres/1B75A8B9-2F00-

4559-9A1F-C55C24A69304/0/MTGRPT_TAG16.pdf

5. Solomon T, Dung NM, Kneen R et al (2002) Seizures and raised

intracranial pressure in Vietnamese patients with Japanese

encephalitis. Brain 125:1084–1093

6. Centers for Disease Control and Prevention (CDC) (2009) Jap-

anese encephalitis among three U.S. Travelers returning from

Asia, 2003–2008. MMWR Morb Mortal Wkly Rep 58:737–740

64 Proc. Natl. Acad. Sci. Sect B. Biol. Sci. (January–March 2012) 82(1):55–68

123

7. Hills SL, Griggs AC, Fischer M (2010) Japanese encephalitis in

travelers from non-endemic countries, 1973–2008. Am J Trop

Med Hyg 82:930–936

8. Marfin AA, Eidex RS, Kozarsky PE et al (2005) Yellow fever

and Japanese encephalitis vaccines: indications and complica-

tions. Infect Dis Clin North Am 19:151–168

9. Fulmali PV, Sapkal GN, Athawale S et al (2011) Introduction of

Japanese encephalitis virus genotype I, India. Emerg Infect Dis

17:319–321

10. Kabilan L, Rajendran R, Arunachalam N et al (2004) Japanese

encephalitis in India: an overview. Indian J Pediatr 71:609–615

11. Saxena V, Dhole TN (2008) Preventive strategies for frequent

outbreaks of Japanese encephalitis in Northern India. J Biosci

33:505–514

12. Miyake M (1964) The pathology of Japanese encephalitis: a

review. Bull World Health Organ 30:153–160

13. Tiroumourougane SV, Raghava P, Srinivasan S (2002) Japanese

viral encephalitis. Postgrad Med J 78:205–215

14. Sucharit S, Surathin K, Shrestha SR (1989) Vectors of Japanese

encephalitis virus (JEV): species complexes of the vectors.

Southeast Asian J Trop Med Public Health 20:611–621

15. Arunachalam N, Samuel PP, Hiriyan J et al (2004) Japanese

encephalitis in Kerala, South India: can Mansonia (Diptera:

Culicidae) play a supplemental role in transmission? J Med

Entomol 41:456–461

16. Rosen L (1986) The natural history of Japanese encephalitis

virus. Annu Rev Microbiol 40:395–414

17. Mall MP, Kumar A, Malik SV (1995) Sero-positivity of

domestic animals against Japanese encephalitis in Bareilly area,

U.P. J Commun Dis 27:242–246

18. Gubler DJ, Rochrig JT (1998) Arboviruses (Togaviridae and

Flavivirdae). In: Collier L, Balows A, Sussman M (eds) Topley

& Wilson’s microbiology and microbial infection, 9th edn.

Arnold, London, pp 579–600

19. Igarashi A, Tanaka M, Morita K (1994) Detection of West Nile

and Japanese encephalitis viral genome sequences in cerebro-

spinal fluid from acute encephalitis cases in Karachi, Pakistan.

In: Takasu T, Ahmed A (eds) Subacute sclerosing panenceph-

alitis: a neuro- viro- patho- epidemio- entomological survey, 1st

edn. Karachi Encephalitis Survey Team Secretariat, Tokyo,

pp 135–142

20. Vijayarani H, Gajanana A (2000) Low rate of Japanese

encephalitis infection in rural children in Thanjavur district

(Tamil Nadu), an area with extensive paddy cultivation. Indian J

Med Res 111:212–214

21. Misra UK, Kalita J (2001) Seizures in Japanese encephalitis.

J Neurol Sci 190:57–60

22. Centers for Disease Control and Prevention (CDC) (2010) Jap-

anese encephalitis vaccines: recommendations of the Advisory

Committee on Immunization Practices (ACIP). MMWR Re-

comm Rep 59(RR-1):1–27

23. Chaturvedi UC, Mathur A, Chandra A, Das SK et al (1980)

Transplacental infection with Japanese encephalitis virus.

J Infect Dis 141:712–715

24. Mathur A, Arora KL, Chaturvedi UC (1981) Congenital infec-

tion of mice with Japanese encephalitis virus. Infect Immun

34:26–29

25. Pealer LN, Marfin AA, Petersen LR et al (2003) Transmission of

West Nile virus through blood transfusion in the United States in

2002. N Engl J Med 49:1236–1245

26. Yongxin Y (1995) Japanese encephalitis in China. Southeast

Asian J Trop Med Public Health 26(suppl 3):17–21

27. Tam N, Yen N (1995) Japanese encephalitis in Vietnam

1985–93. Southeast Asian J Trop Med Public Health 26(suppl

3):47–50

28. Wu YC, Huang YS, Chien LJ et al (1999) The epidemiology of

Japanese encephalitis on Taiwan during 1966–1997. Am J Trop

Med Hyg 61:78–84

29. Lin H, Yang L, Liu Q et al (2011) Time series analysis of

Japanese encephalitis and weather in Linyi City, China. Int J

Public Health 10:1–8

30. Rao PN (2001) Japanese encephalitis. Indian Pediatr 38:

1252–1264

31. Sohn YM (2000) Japanese encephalitis immunization in South

Korea: past, present, and future. Emerg Infect Dis 6:17–24

32. Yamanaka A, Mulyatno KC, Susilowati H et al (2010) Preva-

lence of antibodies to Japanese encephalitis virus among pigs in

Bali and East Java, Indonesia, 2008. Jpn J Infect Dis 63:58–60

33. Shimojima M, Nagao Y, Shimoda H et al (2011) Full genome

sequence and virulence analyses of the recent equine isolate of

Japanese encephalitis virus. J Vet Med Sci 73:813–816

34. Umenai T, Krzysko R, Bektimirov TA et al (1985) Japanese

encephalitis: current worldwide status. Bull World Health Organ

63:625–631

35. Joshi D (1986) Japanese encephalitis in Nepal. JE HFRS Bull

1:5–15

36. Chunsuttiwat S, Warachit P (1995) Japanese encephalitis in

Thailand. Southeast Asian J Trop Med Public Health 26(suppl

3):43–46

37. Bista MB, Shrestha JM (2005) Epidemiological situation of

Japanese encephalitis in Nepal. J Nepal Med Assoc 44:51–56

38. van den Hurk AF, Ritchie SA, Mackenzie JS (2009) Ecology

and geographical expansion of Japanese encephalitis virus.

Annu Rev Entomol 54:17–35

39. Hanna JN, Ritchie SA, Phillips DA et al (1999) Japanese

encephalitis in north Queensland, Australia, 1998. Med J Aust

170:533–536

40. Keiser J, Maltese MF, Erlanger TE et al (2005) Effect of irri-

gated rice agriculture on Japanese encephalitis, including chal-

lenges and opportunities for integrated vector management. Acta

Trop 95:40–57

41. Mathur A, Chaturvedi UC, Tandon HO et al (1982) Japanese

encephalitis epidemic in Uttar Pradesh, India during 1978.

Indian J Med Res 75:161–169

42. Mohan Rao CV, Prasad SR, Rodrigues JJ, Sharma NG et al

(1983) The first laboratory proven outbreak of Japanese

encephalitis in Goa. Indian J Med Res 78:745–750

43. Prasad SR, Kumar V, Marwaha RK et al (1993) An epidemic of

encephalitis in Haryana: serological evidence of Japanese

encephalitis in a few patients. Indian Pediatr 30:905–910

44. Saxena SK, Singh M, Pathak AK, Mathur A (2006) Reply to

‘Encephalitis outbreak finds Indian officials unprepared’. Nat

Med 12:269–270

45. Webb JKG, Pavri K, George S et al (1964) Asian Paediatrics,

the scientific Proceedings of the first all Asian Congress of

Paediatrics. Asia Publishing House, Bombay

46. Namachivayam V, Umayal K (1982) Profile of the 1981 epi-

demic of encephalitis in South Arcot district. In: Proceedings of

the national conference on Japanese encephalitis. ICMR,

pp 30–33

47. Kabilan L, Vrati S, Ramesh S, Srinivasan S et al (2004) Japa-

nese encephalitis virus (JEV) is an important cause of enceph-

alitis among children in Cuddalore district, Tamil Nadu, India.

J Clin Virol 31:153–159

48. Potula R, Badrinath S, Srinivasan S (2003) Japanese encephalitis

in and around Pondicherry, South India: a clinical appraisal and

prognostic indicators for the outcome. J Trop Pediatr 49:48–53

49. Carey DE, Reuben R, Myers RM (1968) Japanese encephalitis

studies in Vellore, South India. I. Virus isolation from mos-

quitoes. Indian J Med Res 56:1309–1318

Proc. Natl. Acad. Sci. Sect B. Biol. Sci. (January–March 2012) 82(1):55–68 65

123

50. Chakravarty SK, Sarkar JK, Chakravarty MS et al (1975) The

first epidemic of Japanese encephalitis studied in India—viro-

logical studies. Indian J Med Res 63:77–82

51. Rathi AK, Kushwaha KP, Singh YD et al (1993) JE virus

encephalitis: 1988 epidemic at Gorakhpur. Indian Pediatr 30:

325–333

52. Reuben R, Gajanana A (1997) Japanese encephalitis in India.

Indian J Pediatr 64:243–251

53. Dhanda V, Thenmozhi V, Kumar NP et al (1997) Virus isolation

from wild-caught mosquitoes during a Japanese encephalitis

outbreak in Kerala in 1996. Indian J Med Res 106:4–6

54. Rao JS, Misra SP, Patanayak SK et al (2000) Japanese

Encephalitis epidemic in Anantapur district, Andhra Pradesh

(October–November, 1999). J Commun Dis 32:306–312

55. Das BP, Lal S, Saxena VK (2004) Outdoor resting preference of

Culex tritaeniorhynchus, the vector of Japanese encephalitis in

Warangal and Karim Nagar districts, Andhra Pradesh. J Vector

Borne Dis 41:32–36

56. Vajpayee A, Dey PN, Chakraborty AK et al (1992) Study of the

outbreak of Japanese encephalitis in Lakhimpur district of

Assam in 1989. J Indian Med Assoc 90:114–115

57. Phukan AC, Borah PK, Mahanta J (2004) Japanese encephalitis

in Assam, northeast India. Southeast Asian J Trop Med Public

Health 35:618–622

58. Gajanana A, Rajendran R, Samuel PP et al (1997) Japanese

encephalitis in south Arcot district, Tamil Nadu, India: a three-

year longitudinal study of vector abundance and infection fre-

quency. J Med Entomol 34:651–659

59. Kar NJ, Bora D, Sharma RC et al (1992) Epidemiological profile

of Japanese encephalitis in Gorakhpur district, Uttar Pradesh,

1982–1988. J Commun Dis 24:145–149

60. Parida M, Dash PK, Tripathi NK et al (2006) Japanese enceph-

alitis outbreak, India, 2005. Emerg Infect Dis 12:1427–1430

61. Saxena SK, Mishra N, Saxena R, Singh M, Mathur A (2009)

Trend of Japanese encephalitis in North India: evidence from

thirty-eight acute encephalitis cases and appraisal of niceties.

J Infect Dev Ctries 3:517–530

62. Sapkal GN, Bondre VP, Fulmali PV et al (2009) Enteroviruses

in patients with acute encephalitis, Uttar Pradesh, India. Emerg

Infect Dis 15:295–298

63. Vaughn DW, Hoke CH Jr (1992) The epidemiology of Japanese

encephalitis: prospects for prevention. Epidemiol Rev 14:

197–221

64. Ludwig GV, Iacono-Connors LC (1993) Insect-transmitted

vertebrate viruses: flaviviridae. In Vitro Cell Dev Biol Anim

29A:296–309

65. Vrati S (2000) Comparison of the genome sequences and the

phylogenetic analyses of the GP78 and the Vellore P20778

isolates of Japanese encephalitis virus from India. J Biosci

25:257–262

66. Chambers TJ, Hahn CS, Galler R et al (1990) Flavivirus genome

organization, expression, and replication. Annu Rev Microbiol

44:649–688

67. Vashist S, Bhullar D, Vrati S (2011) La protein can simulta-

neously bind to both 30- and 50-noncoding regions of Japanese

encephalitis virus genome. DNA Cell Biol 30:339–346

68. Pujhari SK, Prabhakar S, Ratho RK et al (2011) A novel

mutation (S227T) in domain II of the envelope gene of Japanese

encephalitis virus circulating in North India. Epidemiol Infect

139:849–856

69. Solomon T, Ni H, Beasley DW et al (2003) Origin and evolution

of Japanese encephalitis virus in Southeast Asia. J Virol 77:

3091–3098

70. Mackenzie JS, Barrett AD, Deubel V (2002) The Japanese

encephalitis serological group of flaviviruses: a brief introduc-

tion to the group. Curr Top Microbiol Immunol 267:1–10

71. Zhang JS, Zhao QM, Guo XF et al (2011) Isolation, genetic

characteristics of human genotype 1 Japanese encephalitis virus,

China, 2009. PLoS ONE 6:e16418

72. Chen WR, Rico-Hesse R, Tesh RB (1992) A new genotype of

Japanese encephalitis virus from Indonesia. Am J Trop Med

Hyg 47:61–69

73. Uchil PD, Satchidanandam V (2001) Phylogenetic analysis of

Japanese encephalitis virus: envelope gene based analysis

reveals a fifth genotype, geographic clustering, and multiple

introductions of the virus into the Indian subcontinent. Am J

Trop Med Hyg 65:242–251

74. Tsai TF (1997) Factors in the changing epidemiology of Japa-

nese encephalitis and West Nile fever. In: Saluzzo JF, Dodet B

(ed) Factors in the emergence of arbovirus diseases. Elsevier,

Paris, pp 179–189

75. Nabeshima T, Loan HT, Inoue S et al (2009) Evidence of fre-

quent introductions of Japanese encephalitis virus from south-

east Asia and continental east Asia to Japan. J Gen Virol

90:827–832

76. Nabeshima T, Morita K (2010) Phylogenetic analysis of the

migration of Japanese encephalitis virus in Asia. Future Virol

5(3):343–354

77. Huang JH, Lin TH, Teng HJ, Su CL et al (2010) Molecular

epidemiology of Japanese encephalitis virus, Taiwan. Emerg

Infect Dis 16:876–878

78. Yang Y, Ye J, Yang X et al (2011) Japanese encephalitis virus

infection induces changes of mRNA profile of mouse spleen and

brain. Virol J 8:80

79. Shankar SK, Rao TV, Mruthyunjayanna BP et al (1983)

Autopsy study of brains during an epidemic of Japanese

encephalitis in Karnataka. Indian J Med Res 78:431–440

80. Liou ML, Hsu CY (1998) Japanese encephalitis virus is trans-

ported across the cerebral blood vessels by endocytosis in mouse

brain. Cell Tissue Res 293:389–394

81. Das S, Chakraborty S, Basu A (2010) Critical role of lipid rafts in

virus entry and activation of phosphoinositide 30 kinase/Akt sig-

naling during early stages of Japanese encephalitis virus infection

in neural stem/progenitor cells. J Neurochem 115:537–549

82. Desai A, Ravi V, Chandramuki A et al (1995) Proliferative

response of human peripheral blood mononuclear cells to Jap-

anese encephalitis virus. Microbiol Immunol 39:269–273

83. Desai A, Shankar SK, Jayakumar PN et al (1997) Co-existence

of cerebral cysticercosis with Japanese encephalitis: a prognostic

modulator. Epidemiol Infect 118:165–171

84. Ravi V, Parida S, Desai A, Chandramuki A et al (1997) Cor-

relation of tumor necrosis factor levels in the serum and cere-

brospinal fluid with clinical outcome in Japanese encephalitis

patients. J Med Virol 51:132–136

85. Gajanana A, Rajendran R, Thenmozhi V et al (1995) Compar-

ative evaluation of bioassay and ELISA for detection of Japa-

nese encephalitis virus in field collected mosquitos. Southeast

Asian J Trop Med Public Health 26:91–97

86. Mahanta J, Boruah U, Sarmabordoloi JN et al (1996) Japanese

encephalitis virus antibody among normal individuals of Di-

brugarh area, upper Assam. J Commun Dis 28:181–184

87. Kuwayama M, Ito M, Takao S et al (2005) Japanese encephalitis

virus in meningitis patients, Japan. Emerg Infect Dis 11:471–473

88. Chatterjee AK, Banerjee K (1975) Epidemiological studies on

the encephalitis epidemic in Bankura. Indian J Med Res

63:1164–1179

89. Ayukawa R, Fujimoto H, Ayabe M et al (2004) An unexpected

outbreak of Japanese encephalitis in the Chugoku district of

Japan, 2002. Jpn J Infect Dis 57:63–66

90. Wang LH, Fu SH, Wang HY et al (2007) Japanese encephali-

tis outbreak, Yuncheng, China, 2006. Emerg Infect Dis 13:

1123–1125

66 Proc. Natl. Acad. Sci. Sect B. Biol. Sci. (January–March 2012) 82(1):55–68

123

91. Burke DS, Lorsomrudee W, Leake CJ et al (1985) Fatal outcome in

Japanese encephalitis. Am J Trop Med Hyg 34:1203–1210

92. Kumar R, Mathur A, Singh KB et al (1993) Clinical sequelae of

Japanese encephalitis in children. Indian J Med Res 97:9–13

93. Pradhan S, Pandey N, Sashank S et al (1999) Parkinsonism due

to predominant involvement of substantia nigra in Japanese

encephalitis. Neurology 53:1781–1786

94. Gourie Devi M (1984) Clinical aspects and experience in the

management of Japanese encephalitis patients. In: Proceedings

of the national conference of Japanese encephalitis, ICMR, New

Delhi, pp 25–29

95. Misra UK, Kalita J (1997) Movement disorders in Japanese

encephalitis. J Neurol 244:299–303

96. Mathur A, Arora KL, Chaturvedi UC (1982) Transplacental

Japanese encephalitis virus (JEV) infection in mice during

consecutive pregnancies. J Gen Virol 59:213–217

97. Burke DS, Nisalak A, Ussery MA et al (1985) Kinetics of IgM

and IgG responses to Japanese encephalitis virus in human

serum and cerebrospinal fluid. J Infect Dis 151:1093–1099

98. Mathur A, Arora KL, Chaturvedi UC (1983) Host defence

mechanisms against Japanese encephalitis virus infection in

mice. J Gen Virol 64:805–811

99. Mathur A, Arora KL, Rawat S et al (1986) Persistence, latency

and reactivation of Japanese encephalitis virus infection in mice.

J Gen Virol 67:381–385

100. Ravi V, Desai AS, Shenoy PK et al (1993) Persistence of Jap-

anese encephalitis virus in the human nervous system. J Med

Virol 40:326–329

101. Sharma S, Mathur A, Prakash V et al (1991) Japanese enceph-

alitis virus latency in peripheral blood lymphocytes and recur-

rence of infection in children. Clin Exp Immunol 85:85–89

102. Bharadwaj M, Khanna N, Mathur A et al (1991) Effect of

macrophage-derived factor on hypoferraemia induced by Japa-

nese encephalitis virus in mice. Clin Exp Immunol 83:215–218

103. Mathur A, Bharadwaj M, Chaturvedi UC (1990) Alterations in

iron levels in Japanese encephalitis virus infection. Int J Exp

Pathol 71:307–312

104. Johnson RT, Burke DS, Elwell M et al (1985) Japanese

encephalitis: immunocytochemical studies of viral antigen and

inflammatory cells in fatal cases. Ann Neurol 18:567–573

105. Khanna N, Mathur A, Chaturvedi UC (1994) Regulation of vascular

permeability by macrophage-derived chemotactic factor produced

in Japanese encephalitis. Immunol Cell Biol 72:200–204

106. Khanna N, Agnihotri M, Mathur A et al (1991) Neutrophil

chemotactic factor produced by Japanese encephalitis virus

stimulated macrophages. Clin Exp Immunol 86:299–303

107. Khanna N, Mathur A, Bharadwaj M et al (1997) Induction of

hypoglycemia in Japanese encephalitis virus infection: the role

of T lymphocytes. Clin Exp Immunol 107:282–287

108. Tandon A, Singh A, Mathur A et al (2002) Hypoglycemia

during Japanese encephalitis virus infection in man. Int J Exp

Pathol 83:39–43

109. Srivastava S, Khanna N, Saxena SK et al (1999) Degradation of

Japanese encephalitis virus by neutrophils. Int J Exp Pathol

79:17–24

110. Saxena SK, Mathur A, Srivastava RC (2001) Induction of nitric

oxide synthase during Japanese encephalitis virus infection:

evidence of protective role. Arch Biochem Biophys 391:1–7

111. Singh A, Kulshreshtha R, Mathur A (2000) Secretion of the

chemokine interleukin-8 during Japanese encephalitis virus

infection. J Med Microbiol 49:607–612

112. Gupta N, Lomash V, Rao PV (2010) Expression profile of

Japanese encephalitis virus induced neuroinflammation and its

implication in disease severity. J Clin Virol 49:4–10

113. Misra UK, Srivastava R, Kalita J et al (2010) Sequential changes

in serum cytokines and chemokines in a rat model of Japanese

encephalitis. Neuroimmunomodulation 17:411–416

114. Gupta N, Bhaskar AS, Lakshmana Rao PV (2011) Transcriptional

regulation and activation of the mitogen-activated protein kinase

pathway after Japanese encephalitis virus infection in neuro-

blastoma cells. FEMS Immunol Med Microbiol 62:110–121

115. Kumar R, Tripathi P, Singh S, Bannerji G (2006) Clinical fea-

tures in children hospitalized during the 2005 epidemic of Jap-

anese encephalitis in Uttar Pradesh, India. Clin Infect Dis

43:123–131

116. Kalita J, Misra UK (1998) EEG in Japanese encephalitis: a

clinico-radiological correlation. Electroencephalogr Clin Neu-

rophysiol 106:238–243

117. Misra UK, Kalita J, Phadke RV, Wadwekar V et al (2010)

Usefulness of various MRI sequences in the diagnosis of viral

encephalitis. Acta Trop 116:206–211

118. Mathur A, Kumar R, Sharma S et al (1990) Rapid diagnosis of

Japanese encephalitis by immunofluorescent examination of

cerebrospinal fluid. Indian J Med Res 91:1–4

119. Ravi V, Premkumar S, Chandramuki A et al (1989) A reverse

passive haemagglutination test for detection of Japanese

encephalitis virus antigens in cerebrospinal fluid. J Virol

Methods 23:291–298

120. Robinson JS, Featherstone D, Vasanthapuram R et al (2010)

Evaluation of three commercially available Japanese encepha-

litis virus IgM enzyme-linked immunosorbent assays. Am J

Trop Med Hyg 83:1146–1155

121. Lewthwaite P, Shankar MV, Tio PH et al (2010) Evaluation of

two commercially available ELISAs for the diagnosis of Japa-

nese encephalitis applied to field samples. Trop Med Int Health

15:811–818

122. Gould EA, Solomon T, Mackenzie JS (2008) Does antiviral

therapy have a role in the control of Japanese encephalitis?

Antiviral Res 78:140–149

123. Swarup V, Ghosh J, Ghosh S, Saxena A et al (2007) Antiviral

and anti-inflammatory effects of rosmarinic acid in an experi-

mental murine model of Japanese encephalitis. Antimicrob

Agents Chemother 51:3367–3370

124. Saxena SK, Mathur A, Srivastava RC (2003) Inhibition of

Japanese encephalitis virus infection by diethyldithiocarbamate

is independent of its antioxidant potential. Antivir Chem Che-

mother 14:91–98

125. Nazmi A, Dutta K, Basu A (2010) Antiviral and neuroprotective

role of octaguanidinium dendrimer-conjugated morpholino

oligomers in Japanese encephalitis. PLoS Negl Trop Dis 4:e892

126. Sebastian L, Madhusudana SN, Ravi V et al (2011) Myco-

phenolic acid inhibits replication of Japanese encephalitis virus.

Chemotherapy 57:56–61

127. Dung NM, Turtle L, Chong WK et al (2009) An evaluation of

the usefulness of neuroimaging for the diagnosis of Japanese

encephalitis. J Neurol 256:2052–2060

128. Japanese Encephalitis Fact Sheet. Centre for Disease Control and

Prevention. National Centre for Emerging and Zoonotic Infec-

tious Diseases (NCEZID). Division of Vector-Borne Diseases

(DVBD). www.cdc.gov/ncidod/dvbid/jencephalitis/facts.htm

129. Chambers TJ, Tsai TF, Pervikov Y et al (1997) Vaccine

development against dengue and Japanese encephalitis: report of

a World Health Organization meeting. Vaccine 15:1494–1502

130. World Health Organization Third Biregional Meeting on Control

of Japanese encephalitis; 2007 Apr 26–27; Ho Chi Minh City,

Vietnam. Available from http://www.wpro.who.int/NR/rdonlyres/

50129D1D-E9B3-4707-A62E-0A541DBC3032/0/MTGRPT_

JEBireg3.pdf

Proc. Natl. Acad. Sci. Sect B. Biol. Sci. (January–March 2012) 82(1):55–68 67

123

131. Hoke CH, Nisalak A, Sangawhipa N et al (1988) Protection

against Japanese encephalitis by inactivated vaccines. N Engl J

Med 319:608–614

132. Bharati K, Vrati S (2006) Japanese encephalitis: development of

new candidate vaccines. Expert Rev Anti Infect Ther 4:313–324

133. Plesner AM (2003) Allergic reactions to Japanese encephalitis

vaccine. Immunol Allergy Clin North Am 23:665–697

134. Tsai TF, Chang GJ, Yu XY (1999) Japanese encephalitis vac-

cine. In: Plotkins SA, Orenstein OK (eds) Vaccine, vol 18.

Thailand, 13–15 October, 1998, pp 1–25

135. Srivastava AK, Putnak JR, Lee SH et al (2001) A purified

inactivated Japanese encephalitis virus vaccine made in Vero

cells. Vaccine 19:4557–4565

136. Appaiahgari MB, Vrati S (2004) Immunogenicity and protective

efficacy in mice of a formaldehyde-inactivated Indian strain of

Japanese encephalitis virus grown in Vero cells. Vaccine

22:3669–3675

137. Tandan JB, Ohrr H, Sohn YM, Yoksan S et al (2007) Single

dose of SA 14-14-2 vaccine provides long-term protection

against Japanese encephalitis: a case-control study in Nepalese

children 5 years after immunization. Vaccine 25:5041–5045

138. WHO (2006) Japanese encephalitis vaccines. Wkly Epidemiol

Rec 81:331–340

139. Appaiahgari MB, Vrati S (2010) IMOJEV(�): a Yellow fever

virus-based novel Japanese encephalitis vaccine. Expert Rev

Vaccines 9:1371–1384

140. Beasley DW, Lewthwaite P, Solomon T (2008) Current use and

development of vaccines for Japanese encephalitis. Expert Opin

Biol Ther 8:95–106

141. Seif SA, Morita K, Igarashi A (1996) A 27 amino acid coding

region of JE virus E protein expressed in E. coli as fusion protein

with glutathione-S-transferase elicit neutralizing antibody in

mice. Virus Res 43:91–96

142. Appaiahgari MB, Saini M, Rauthan M et al (2006) Immuniza-

tion with recombinant adenovirus synthesizing the secretory

form of Japanese encephalitis virus envelope protein protects

adenovirus-exposed mice against lethal encephalitis. Microbes

Infect 8:92–104

143. Bharati K, Rani R, Vrati S (2009) Evaluation of Japanese

encephalitis virus DNA vaccine candidates in rhesus monkeys

[Macaca mulatta]. Vaccine 27:10–16

144. Appaiahgari MB, Vrati S (2007) DNAzyme-mediated inhibition

of Japanese encephalitis virus replication in mouse brain. Mol

Ther 15:1593–1599

145. Elias C, Okwo-Bele JM, Fischer M (2009) A strategic plan for

Japanese encephalitis control by 2015. Lancet Infect Dis 9:7

146. Caramello P, Canta F, Balbiano R et al (2007) A case of

imported JE acquired during short travel in Vietnam. Are current

recommendations about vaccination broader? J Travel Med

14:346–348

147. Zhang S, Yin Z, Suraratdecha C et al (2011) Knowledge, atti-

tudes and practices of caregivers regarding Japanese encephalitis

in Shaanxi Province, China. Public Health 125:79–83

68 Proc. Natl. Acad. Sci. Sect B. Biol. Sci. (January–March 2012) 82(1):55–68

123